The invention relates generally to the field of viral vectors useful in gene delivery, and more particularly, to recombinant adeno-associated viruses (rAAV).
Adeno-associated viruses are small, single-stranded DNA viruses which require helper virus to facilitate efficient replication (K. I. Berns, Parvoviridae: the viruses and their replication, p. 1007–1041, in F. N. Fields et al., FUNDAMENTAL VIROLOGY, 3rd ed., vol. 2, (Lippencott-Raven Publishers, Philadelphia, Pa.) (1995)). The 4.7 kb genome of AAV is characterized by two inverted terminal repeats (ITR) and two open reading frames which encode the Rep proteins and Cap proteins, respectively. The Rep reading frame encodes four proteins of molecular weight 78 kD, 68 kD, 52 kD and 40 kD. These proteins function mainly in regulating AAV replication and integration of the AAV into a host cell's chromosomes. The Cap reading frame encodes three structural proteins of molecular weight 85 kD (VP 1), 72 kD (VP2) and 61 kD (VP3) (Berns, cited above). More than 80% of total proteins in AAV virion comprise VP3. The two ITRs are the only cis elements essential for AAV replication, packaging and integration. There are two conformations of AAV ITRs called “flip” and “flop”. These differences in conformation originated from the replication model of adeno-associated virus which uses the ITR to initiate and re-initiate the replication (R. O. Snyder et al., J. VIROL., 67:6096–6104 (1993); K. I. Berns, MICROBIOLOGICAL REVIEWS, 54:316–329 (1990)).
AAVs have been found in many animal species, including primates, canine, fowl and human (F. A. Murphy et al., “The Classification and Nomenclature of Viruses: Sixth Report of the International Committee on Taxonomy of Viruses”, ARCHIVES OF VIROLOGY, (Springer-Verlag, Vienna) (1995)). Six primate serotypes have been reported (AAV1, AAV2, AAV3, AAV4, AAV5 and AAV6). With the exception of AAV5, which was isolated from a patient's lesion, all other AAV serotypes were discovered in cell culture. In addition, AAV5 is the only serotype which does not show cross-complementation with any other serotype. Whereas the homology among the other serotypes is in the range of 75% to 82%, AAV5 reveals only about 50% homology with other AAV serotypes on the protein level. The sequence of the AAV5 has been cloned and sequenced, and recombinant AAV5 particles have been generated (J. A. Chiorini et al, J. VIROL., 73(2):1309–1319 (February 1999)). The authors of this paper also reported that an attempt to pseudotype an AAV2 vector in an AAV5 capsid and an attempt to pseudotype an AAV5 vector in an AAV2 capsid were unsuccessful.
A number of unique properties make AAV a promising vector for human gene therapy (Muzyczka, CURRENT TOPIC IN MICROBIOLOGY AND IMMUNOLOGY, 158:97–129 (1992)). Unlike other viral vectors, AAVs have not been shown to be associated with any known human disease and are generally not considered pathogenic. Wild type AAV is capable of integrating into host chromosomes in a site specific manner (R. M. Kotin et al., PROC. NATL. ACAD. SCI, USA, 87:2211–2215 (1990); R. J. Samulski, EMBO J., 10(12):3941–3950 (1991)). Recombinant AAV vectors can integrate into tissue cultured cells in chromosome 19 if the rep proteins are supplied in trans (C. Balague et al., J. VIROL., 71:3299–3306 (1997); R. T. Surosky et al., J. VIROL., 71:7951–7959 (1997)). The integrated genomes of AAV have been shown to allow long term gene expression in a number of tissues, including muscle, liver, and brain (K. J. Fisher, NATURE MED., 3(3):306–312 (1997); R. O. Snyder et al., NATURE GENETICS, 16:270–276 (1997); X. Xiao et al., EXPERIMENTAL NEUROLOGY, 144:113–124 (1997); Xiao, J. VIROL., 70(11):8098–8108 (1996)).
AAV2 has been shown to be present in about 80–90% of the human population. Earlier studies showed that neutralizing antibodies for AAV2 are prevalent (W. P. Parks et al., J. VIROL., 2:716–722 (1970)). The presence of such antibodies may significantly decrease the usefulness of AAV vectors based on AAV2 despite its other merits.
What are needed in the art are vectors characterized by the advantages of AAV-2, such as those described above, without the disadvantages, e.g., the presence of destructive neutralizing antibodies.